Structural Biology

Over the years since NMR was first applied to solve problems in structural biology, it has undergonedramaticdevelopmentsinbothNMRinstrumenthardwareandmethodology. While it is established that NMR is one of the most powerful tools for understanding biological p- cesses at the atomic level, it has become increasingly difficult for authors and instructors to make valid decisions concerning the content and level for a graduate course of NMR in str- turalbiology. BecausemanyofthedetailsinpracticalNMRarenotdocumentedsystematically, students entering the field have to learn the experiments and methods through communication with other experienced students or experts. Often such a learning process is incomplete and unsystematic. This book is meant to be not only a textbook, but also a handbook for those who routinely use NMR to study various biological systems. Thus, the book is organized with experimentalists in mind, whether they are instructors or students. For those who have a little or no background in NMR structural biology, it is hoped that this book will provide sufficient perspective and insight. Those who are already experienced in NMR research may find new information or different methods that are useful to their research. Because understanding fundamental principles and concepts of NMR spectroscopy is essential for the application of NMR methods to research projects, the book begins with an introduction to basic NMR principles. While detailed mathematics and quantum mechanics dealing with NMR theory have been addressed in several well-known NMR books, Chapter 1 illustrates some of the fundamental principles and concepts of NMR spectroscopy in a more descriptiveandstraightforwardmanner.

Contenu

PREFACE.- CHAPTER 1 BASIC PRINCIPLES OF NMR: Introduction.- Nuclear Spin in a Static Magnetic Field.- Rotating Frame.- Bloch Equations.- Fourier Transformation and its Applications in NMR.- Nyquist Theorem and Digital Filters.- Chemical Shift.- Nuclear Coupling.- Nuclear Overhauser Effect.- Relaxation.- Selection of Coherence Transfer Pathways.- Approaches to Understanding NMR Experiments.- Questions.- Appendix A: Product Operators.- Uncoupled Spins.- A2. Two Coupled Spins.- References.- CHAPTER 2 INSTRUMENTATION: System Overview.- Magnet.- Transmitter.- Receiver.- Probe.- Quarter-wavelength Cable.- Analog/Digital Converters.- Instrument specifications.- Test or Measurement Equipment.- Spectrum Analyzer.- System Noise Measurement.- Questions.- References.- CHAPTER 3 NMR SAMPLE PREPARATION: Introduction.- Expression Systems.- Overexpression of Isotope-Labeled Proteins.- Purification of Isotope-Labeled Proteins.- NMR Sample Preparation.- Examples of Protocols for Preparing 15N/13C Labeled Proteins.- Questions.- References.- CHAPTER 4 PRACTICAL ASPECTS: Tuning the Probe.- Shimming and Locking.- Instrument Calibrations.- Selective Excitation with Narrow Band and Off-Resonance Shape Pulses.- Composite Pulses.- Adiabatic Pulses.- Pulsed Field Gradients.- Solvent Suppression.- NMR Data Processing.- Two-Dimensional Experiments.- Questions.- References.- CHAPTER 5 MULTIDIMENSIONAL HETERONUCLEAR NMR EXPERIMENTS: Two-dimensional heteronuclear experiments.- Overview of Triple-Resonance Experiments.- General Procedure of Setup and Data Processing for 3D Experiments.- Experiments for Backbone Assignments.- Experiments for Side-Chain Assignment.- 3D Isotope-Edited Experiments.- Sequence-Specific Resonance Assignments of Proteins.- Assignment of NOE Cross-Peaks.- Questions.- References.- CHAPTER 6 STUDIES OF SMALL BIOLOGICAL MOLECULES: Ligand-protein Complexes.- Study of Metabolic Pathways by NMR.- Questions.- References.- CHAPTER 7 PROTEIN STRUCTURE DETERMINATION FROM NMR DATA: Introduction and Historical Overview.- NMR Structure Calculation Methods.- NMR Parameters for Structure Calculation.- Preliminary Secondary Structural Analysis.- Tertiary Structure Determination.- Protein Complexes.- Questions.- Appendix B1. Sa.inp-XPLOR Protocol for Protein Structure Calculation.- Appendix B2. Example of NOE Table.- Appendix B3. Example of Dihedral Angle Restraint Table.- Appendix B4. Example of Chemical Shift Table for TALOS.- Appendix B5. Example of Hydrogen Bond Table.- Appendix B6. Example of Input File to Generate a Random-Coil Coordinates.- Appendix B7. Example of Input File to Generate a Geometric PSF File.- References.- CHAPTER 8 PROTEIN DYNAMICS: Theory of Spin Relaxation in Proteins.- Experiments for Measurements of Relaxation Parameters.- Relaxation Data Analysis.- Questions.- References.- MULTIPLE-CHOICE QUESTIONS.- ANSWERS TO MULTIPLE CHOICE QUESTIONS.- NOMENCLATURE AND SYMBOLS.- INDEX.